The cellular and subcellular movement of calcium (Ca2+) in heart cells underlies cellular contraction and influences electrical behavior. The PI and his co-mentors have recently discovered new features of Ca2+ movement in heart cells that have profound implications for understanding heart function. Here, the PI proposes to investigate the novel discovery of "invisible Ca2+ leak" by combining quantitative mathematical investigations with experimental tests. Ca2+ leak is the loss of Ca2+ from intracellular storage organelles and plays a vital role in maintaining healthy cellular Ca2+ content by balancing uptake from the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump. Understanding Ca2+ leak and its molecular basis is essential for experimental and theoretical examination of cellular physiology, pathophysiology (including heart failure and arrhythmias), and developing new therapeutics. The planned investigation of SR Ca2+ leak will exploit novel and very efficient mathematical tools recently invented by the PI and his co-mentors that will enable a fully stochastic mathematical investigation of the Ca2+ signaling in single cardiac ventricular myocytes. Work by the PI and his co-mentors has provided preliminary mathematical and biological characterization of two components of Ca2+ leak: Ca2+ sparks (see introduction) and "invisible," non-spark Ca2+ leak. These two components appear to play a role in both normal and arrhythmogenic Ca2+ signaling behavior but have yet to be characterized at the molecular level. The proposed work will test this critical Ca2+ signaling behavior by combining mathematical modeling investigations with single mouse ventricular myocyte experiments. Confocal Ca2+ imaging with simultaneous patch clamp experiments will be carried out in enzymatically dissociated cells to inform the modeling and test the findings. Ca2+ sparks, [Ca2+]i transients, and membrane currents will be investigated in myocytes from control mice (C57BL/6) and from mutant mice with specific alterations in the cardiac ryanodine receptor (RyR2) (C57BL/6- R2474S) that produce Ca2+-dependent arrhythmias (see [1]). Preliminary work by the PI and his co-mentors suggest that there are profound differences in the Ca2+ leak characteristics in the control and mutant heart cells. The proposed investigation into SR Ca2+ leak in heart seeks to address two critically important questions on cardiac Ca2+ signaling: 1) What is the molecular mechanism of SR Ca2+ leak in healthy myocytes? and 2) how do arrhythmogenic RyR2 mutations affect SR Ca2+ leak? The unique feature of the proposed work is the combination of modeling and experiments in a richly interactive environment with a strong record of success in such work. For the PI, the investigation nicely supports his long-term plan to combine theoretical investigations with practical and informative tests with the prospect of broadening our understanding of cardiac cellular function. PUBLIC HEALTH RELEVANCE: The research will explore the molecular mechanisms responsible for calcium leak in mammalian heart cells. The movement of intracellular calcium underlies cellular contraction, and alterations in calcium handling can change the intracellular calcium balance, leading to pathological conditions such as arrhythmias and even heart failure. Understanding calcium leak and its molecular basis will provide insights into cellular physiology and identify avenues for developing new therapeutics.